Methods for the humanization of antibodies and humanized antibodies thereby obtained

ABSTRACT

Method for the humanization of the VH and VL variable regions of an animal antibody of known sequence, humanized animal antibody obtainable according to the method, in particular anti-NGF and anti-TrkA humanized animal antibodies.

RELATED APPLICATIONS

The present application is filed as a continuation application of U.S.patent application Ser. No. 12/838,062, which was filed on Jul. 16,2010, which was filed as a divisional of U.S. patent application Ser.No. 10/583,618, which was filed on Sep. 19, 2008 (now U.S. Pat. No.8,296,079), which was filed as a National Stage Entry of InternationalPatent Application No. PCT/IT04/00722, which was filed on Dec. 23, 2004,which claims benefit of Italian Patent Application No. RM2003000601,which was filed on Dec. 24, 2003. The entire text of the aforementionedapplications is incorporated herein by reference in its entirety.

BACKGROUND

The present invention relates to a method for the humanization ofantibodies, by means of determining and comparing three-dimensionalstructures, humanized antibodies thereby obtained and their uses intherapy and diagnostics in vivo.

The therapeutic and diagnostic application of monoclonal antibodies ofanimal origins in humans has fundamental contraindications especiallyfor therapeutic regimes which necessitate for repeated administrations.In particular, murine monoclonal antibodies have a relatively shorthalf-life and, when used in humans, lack some fundamental functionalcharacteristics of immunoglobulins, such as complement-dependentcytotoxicity and cell-mediated cytotoxicity.

Moreover, monoclonal antibodies of non-human origin contain immunogenicamino acid sequences if injected into patients. Numerous studies haveshown that after the injection of an exogenous antibody, subjectsdevelop a rather strong immune reaction against the antibody itself(known as HAMA—human anti-mouse antibodies—reaction), completelyeliminating its therapeutic usefulness, with the formation ofimmunocomplexes, alteration of pharmacokinetics, production of allergicreactions, etc. Moreover, considering the growing number of differentmonoclonal antibodies developed in mice or in other mammals (and thusantigenic for humans) for the therapy of different pathologies,treatments, also for non correlated therapies can be ineffective or evendangerous due to cross-reactivity. Although the production of so-calledchimeric antibodies (variable murine regions joined to constant regionsof human origin) has yielded some positive result, a significantimmunogenicity problem still remains.

Humanized antibodies have at least three potential advantages withrespect to antibodies of animal origin in the field of therapeutic usein humans. In the first place, the effector region, being human, canbetter interact with the other parts of the human immune system,destroying target cells more efficiently by means ofcomplement-dependent cytotoxicity, or cell-mediated, antibody dependentcytotoxicity. Moreover, the human immune system does not recognize theframework or the constant region (C) of the humanized antibody asexogenous, and hence the antibody response against the humanizedantibody is minimized, both relative to that against a murine antibody(totally extraneous) and relative to the response induced by a chimericantibody (partially extraneous).

It has been reported that murine antibodies injected into humans have amuch shorter half-life time than normal antibodies (Shaw et al., 1987).Humanized antibodies have a very similar half life to that of naturalhuman antibodies, allowing less frequent administration and lower doses.

The basic principle of humanization is configured in transferring thespecificity of antigen recognition, i.e. the CDR domains, in the contextof a human immunoglobulin (“CDR grafting”, Winter and Milstein, 1991).Several examples of humanized antibodies, produced in the attempt tosolve the problem of immunogenicity, have been reported (Maeda et al.,1991; Singer et al., 1993; Tempest et al., 1994; Kettleborough et al.,1991; Hsiao et al., 1994; Baca et al., 1997; Leger et al., 1997; Elliset al., 1995; Sato et al., 1994; Jones et al., 1986; Benhar et al.,1994; Sha and Xiang, 1994; Shearman et al., 1991; Rosok et al., 1996;Gussow & Seemann, 1991; Couto et al., 1994; Kashmiri et al., 1995; Bakeret al., 1994; Riechmann et al., 1988; Gorman et al., 1991; Verhoeyen etal., 1988; Foote & Winter, 1992; Lewis & Crowe, 1991; Co et al., 1991;Co et al., 1991; Verhoeyen et al., 1991; Eigenbrot et al., 1994;Hamilton et al., 1997; Tempest et al., 1995; Verhoeyen et al., 1993;Cook et al., 1996; Poul et al., 1995; Co et al., 1992; Graziano et al.,1995; Presta et al., 1993; Hakimi et al., 1993; Roguska et al., 1996;Adair et al., 1994; Sato et al., 1993; Tempest et al., 1991; Sato etal., 1996; Kolbinger et al., 1993; Zhu and Carter, 1995; Sims et al.,1993; Routledge et al., 1991; Roguska et al., 1994; Queen et al., 1989;Carter et al., 1992).

The transcription of an antibody from animal (generally murine) tohumanized entails the compromise between opposite requirements, whosesolution varies case by case. To minimize immunogenicity, immunoglobulinshall maintain as much of the accepting human sequence as possible. Inany case, to preserve the original binding properties, theimmunoglobulin framework should contain a sufficient number of mutationsin the accepting human sequence to guarantee that the conformation ofthe CDR regions is as similar as possible to that in the donor murineimmunoglobulin. As a consequence of these opposite considerations, formany humanized antibodies a significant loss in binding affinity withrespect to the corresponding murine antibodies has been reported (Joneset al., 1986; Shearman et al., 1991; Kettleborough, 1991; Gorman et al.,1991; Riechmann et al., 1988).

Currently, the most common method for the production of humanizedimmunoglobulin is based on the use of appropriate genomic, syntheticsequences, as well as cDNA (Reichmann et al., 1988).

The patent application EP 592106 discloses a method for the humanizationof antibodies from rodents. The method is based on the identification ofthe amino acid residues exposed at the surface of the three-dimensionalstructure of the antibody to be humanized, on the identification of theamino acid residues in the same positions on the corresponding humanantibody, and on the replacement of the residues identified in thesequence of the rodent antibody with those identified in the humanantibody.

SUMMARY OF THE INVENTION

The authors of the present invention set up a method to obtain optimizedhumanized forms of immunoglobulins which are substantially notimmunogenic in humans, with an approach that is consistently based onstructural data, obtained experimentally, deriving from crystallographicstudies. The method of the invention allows one to obtain antibodies ina form adapted to therapeutic formulation and to other medical anddiagnostic applications.

The invention relates to a method fully based on structural data toconduct the first design stages (generally more subject to error) ofhumanization. Humanized immunoglobulins have two pairs of heterodimersbetween light and heavy chain, with at least one of the chains bearingone or more CDRs of animal origin, functionally bound to segments ofregions of the framework of human origin. For example, CDRs of animalorigin, together with amino acid residues, naturally associated, also ofanimal origins, are introduced in framework regions of human origin, toproduce humanized immunoglobulins able to bind the respective antigens,with affinities comparable to the affinities of the originalimmunoglobulins of animal origin.

The method of the invention led to humanized antibodies suitable fortherapeutic and diagnostic applications. In particular, humanizedimmunoglobulins have been obtained, derived from anti-TrkA antibodies(Patent EP 1181318) and from anti-NGF antibodies able to bind with highspecificity respectively TrkA and NGF, neutralizing the interactionbetween ligand and receptors. Such molecules are useful for thetreatment of tumors which depend on NGF/TrkA, of chronic pain and ofinflammatory forms, and for diagnostic purposes, for in vivo imaging,e.g. on TrkA positive tumors, or on basal forebrain as a precociousmarker of Alzheimer's Disease. In particular, humanized anti-TrkAantibodies find specific therapeutic and diagnostic application in theinflammatory forms of the urinary tract and of the pelvic region. Inparticular, humanized anti-NGF antibodies find specific therapeutic anddiagnostic application in pathologies induced by HIV virus, to induceapoptosis of immune cells, such as HIV infected, NGF dependentmacrophages.

Therefore, an object of the present invention is to provide a method forthe humanization of the VH and VL variable regions of a animal antibodyof known sequence, comprising the steps of:

-   a) if not available, obtaining the crystallographic structure of the    VH and VL regions of the animal antibody;-   b) pre-selecting a series of 0 to n possible frameworks acceptors of    human origin or humanized antibodies, whose structure was determined    experimentally with a resolution of no less than 3 Å, based on the    highest level of homology and identity with the primary sequence of    the framework of the animal antibody;-   c) conducting a structural comparison between the VH and VL variable    regions of the animal antibody and the regions VH and VL obtained in    b), respectively and calculating for each comparison the RMS, to    identify the region VH and the region VL of human origin with the    smaller RMS;-   d) inserting in appropriate position the sequences of the regions    CDR of the animal antibody in the human sequences identified in c);-   e) if necessary, retromutate one or more amino acid residues of the    human VH and VL regions identified in c).

Preferably, the modifications of the antibody take place withrecombining DNA techniques.

In a preferred embodiment, the animal antibody is an anti-NGF antibody,preferably it is the alpha D11 antibody, and the humanized sequencesessentially have the following VH sequences:

Hum alpha D11 (SEQ ID No. 17)VH,EVQLVESGGGLVQPGGSLRLSCAASGFSLTNNNVNWVRQAPGKGLEWVGGVWAGGATDYNSALKSRFTISRDNSKNTAYLQMNSLRAEDTAVYYCARDGGYSSSTLYAMDAWGQGTLVTVSS, and VL: Hum alpha D11Vk, (SEQ ID No. 18)DIQMTQSPSSLSASVGDRVTITCRASEDIYNALAWYQQKPGKAPKLLIYNTDTLHTGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQHYFHYPR TFGQGTKVEIK.

In an alternative embodiment, the animal antibody is an anti-TrkAantibody, preferably it is the alpha MNAC13 antibody, and the humanizedsequences essentially have the following sequences:

VH: HumMNAC13VH, (SEQ ID No. 37)EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYTMSWARQAPGKGLEWVAYISKGGGSTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDSAVYYCARGAMFGNDFFFPMDRWGQGTLVTVSSA, and VL: Hum MNAC13Vk, (SEQ ID No. 38)DIVLTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKPGQAPKLLIYTTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCHQWSSYPWT FGGGTKVEIK.

The humanized immunoglobulins of the present invention (or derivedfragments which maintain binding activities and other compounds whichcan be derived) can be produced by means of known recombining DNAtechniques. As a function of the subsequent use of the humanizedimmunoglobulins, transgenic animals or transfected cells can be used fortheir expression, preferably immortalized eukaryotic cells (such asmyeloma or hybridoma cells), but also prokaryotic hosts, insect orvegetable cells. The coding polynucleotides for the resulting sequencesof the humanized immunoglobulins can also be obtained by synthesis.

The humanized immunoglobulins of the present invention can be used aloneor in combination with other therapeutic agents. In case of use asanti-tumor agents, a chemotherapeutic agent will be preferred, which mayvary depending on the pharmacological application (such as anthracyclin,paclitaxel, cisplatin, gemcytabin, non steroidal and corticosteroidanti-inflammatory drugs, or immunosuppressants), as well as with alldrugs currently applied in the therapy of each specific pathology.Humanized immunoglobulins or their complexes can be prepared in the formof pharmacologically acceptable dosages, which vary depending on thetype of administration.

Definitions

The term “substantially identical” within the context of twopolynucleotides or polypeptides (respectively sequences of coding DNAfor humanized immunoglobulins or amino acid sequences of humanizedimmunoglobulins, or portions thereof) refers to two or more sequenceswhich have a minimum of 80% (preferably 90-95% or more) of identity inthe nucleotide or amino acid residues, when compared and aligned withmaximum correspondence. Generally, the “substantial identity” isverified in regions that are at least 50 residues long, more preferablyon a region of at least 100 residues or, in optimal conditions, on over150 residues or on the complete sequences. As described below, any twosequences of antibodies can be aligned in only one way, using Kabat'snumbering scheme. Consequently, for antibodies the percentage ofidentity has a unique and well defined meaning. The amino acids of thevariable regions of the heavy and light chains of mature immunoglobulinsare designated Hx and Lx, with x being the number that designates theposition of the amino acid according to Kabat's numbering scheme,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda Md., 1987, 1991). Kabat has determined a list of aminoacid sequences of antibodies for each subgroup as well as a list of themost frequent amino acids in each position in each subgroup to generatea consensus sequence. Kabat uses a method to assign a number to eachamino acid of each sequence in the list and this method for assigningthe number of each residue has become a standard method in the field.Kabat's scheme can be extended to other antibodies not present in hisstudy, aligning the antibody in question with one of the consensussequences identified by Kabat, basing on the preserved amino acids. Useof Kabat's numbering scheme allows one to easily identify the aminoacids in equivalent positions in different antibodies. For example, anamino acid in L10 position in an antibody of human origin occupies theequivalent position of an amino acid in L10 position in an antibody ofmurine origin.

It is well known that the basic structural unit of an antibody comprisesa tetramer. Each tetramer is constituted by two identical pairs ofpolypeptide chains, each of which is composed by a light chain (25 KDa)and by a heavy chain (50-75 KDa). The amino-terminal region of eachchain includes a variable region of about 100-110 or more amino acids,which is involved in antigen recognition. The carboxy-terminal region ofeach chain comprises the constant region that mediates the effectorfunction. The variable regions of each pair of light and heavy chainsform the binding site of the antibody. Therefore, an intact antibody hastwo binding sites.

Light chains are classified as γ or λ. Heavy chains are classified as γ,μ, α, ε and they define the isotype of the antibody as respectively IgG,IgM, IgA, IgD, or IgE. Inside both the light and the heavy chain, thevariable and constant regions are joined by a “J” region of about 12amino acids or more, whilst only the heavy chains include a “D” regionof about 10 amino acids (Paul, 1993).

The variable regions of each pair of light and heavy chains form thebinding site of the antibody. They are characterized by the same generalstructure constituted by relatively preserved regions called frameworks(FR) joined by three hyper-variable regions called complementaritydetermining regions (CDR) (Kabat et al., 1987; Chothia and Lesk, 1987).The CDRs of the two chains of each pair are aligned by the frameworkregions, acquiring the function of binding a specific epitope. Startingfrom the amino-terminal region towards the carboxy-terminal region, thevariable domains both of the light chain and of the heavy chain compriseand alternation of FR and CDR regions: FR, CDR, FR, CDR, FR, CDR, FR;consequently, both the heavy chain and the light chain are characterizedby three CDRs, respectively CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3.Amino acid assignment to each region was conducted according to thedefinitions by Kabat (1987 and 1991) and/or Chothia & Lesk (1987),Chothia et al. (1989).

Preferably, the analogs of the exemplified humanized immunoglobulinsdiffer from the original immunoglobulins due to conservative amino acidsubstitutions. In order to classify the amino acid substitutions asconservative or non conservative, amino acids can be grouped as follows:

-   Group I (hydrophobic lateral chains): M, A, V, L, I;-   Group II (neutral hydrophilic lateral chains): C, S, T, N, Q;-   Group III (acid lateral chains): D, E;-   Group IV (basic lateral chains): K, R;-   Group V (residues that influence the orientation of the main chain):    G, P;-   Group VI (aromatic lateral chains): F, Y, W.

Conservative amino acid substitutions are substitutions between aminoacid of the same class, whilst non conservative amino acid substitutionsentail an exchange between members of different classes.

The term “epitope” includes every protein determinant able to bind animmunoglobulin in specific fashion. Generally, epitopes are formed bysets of chemically active surfaces of macromolecules, such as lateralchains of amino acid or sugars and they generally have specificchemical-physical and conformational characteristics.

The term “immunoglobulins” refers to proteins which consist of one ormore polypeptides coded by genes of the immunoglobulins. Immunoglobulinscan exist in a variety of forms, in addition to the tetramer antibodyform: for example, they include fragments Fv, Fab e F(ab′) as well asbifunctional hybrid antibodies (Lanzavecchia et al., 1987) and singlechain Fv fragments (Hood et al., 1984; Harlow and Lane, 1988;Hunkapiller and Hood, 1986).

Chimeric antibodies are antibodies whose genes for the light and heavychains have been engineering starting from gene regions ofimmunoglobulins belonging to different species. For example, variablesegments (V) of the genes of a monoclonal mouse antibody can be joinedto constant segments (C) of an antibody of human origin. A therapeuticchimeric antibody, therefore, is a hybrid protein which consists of thedomain V which recognizes the antigen deriving from a mouse antibody andin the effector domain C deriving from a human antibody (although othercombinations of mammal species can be used).

The term “framework” refers to those portions of the variable regions ofthe light and heavy chain of the immunoglobulins that are relativelypreserved (not belonging to the CDRs) between different immunoglobulinswithin a species, according to Kabat's definition. Hence, a humanframework is a framework region that is substantially identical (atleast 85% or more) to the framework that is naturally found in humanantibodies.

The term “humanized immunoglobulin” refers to an immunoglobulin whichcomprises a human framework and at least one CDR deriving from a nonhuman antibody and in which each constant region present issubstantially identical to a region of human immunoglobulin (at least85%, preferably at least 90-95% identical). Hence, all the parts of ahumanized immunoglobulin except the CDR are substantially identical tothe corresponding regions of one or more sequences of natural humanimmunoglobulins. For example, the chimeric antibodies, constituted byvariable mouse regions and constant regions of human origin, are notincluded among the humanized immunoglobulins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the analysis by means of polyacrylamide in denaturingconditions (SDS-PAGE 12%) and coloring with Coomassie Blue of the resultof the purification of the Fab fragment of the MNAC13 antibody (well 1:sample of MNAC13 antibody digested proteolytically with papaine; well 2:fraction bound to the DEAE Sephacell ionic exchange resin and elutedwith NaCl 250 mM; well 3: molecular weights; well 4: Fab fragment of thepurified and concentrated MNAC13 antibody); FIG. 1B depicts a typicalcrystal of the Fab fragment of the MNAC13 antibody FIG. 1C depicts ahigh resolution diffraction spectrum obtained with a crystal of the Fabfragment of the MNAC13 antibody; FIG. 1D is a Ramachandran chart of thetorsion angles of the main chain of the heavy and light domains of theFab fragment of the MNAC13 antibody.

FIG. 2A depicts the analysis by means of polyacrylamide in denaturingconditions (SDS-PAGE 12%) and coloring with Coomassie Blue of the resultof the purification of the Fab fragment of the αD11 antibody (well 1:sample of αD11 antibody digested proteolytically with papaine; well 2:Fab fragment of the purified and concentrated αD11 antibody; well 3:molecular weights); FIG. 2B depicts a typical crystal of the Fabfragment of the αD11 antibody FIG. 2C depicts a high resolutiondiffraction spectrum obtained with a crystal of the Fab fragment of theαD11 antibody; FIG. 2D is a Ramachandran chart of the torsion angles ofthe main chain of the heavy and light domains of the Fab fragment of theαD11 antibody.

FIGS. 3A through 3D depict distributions of the humanized or humanorigin antibodies (named using the PDB codes of their crystallographicstructures) according to the three analyzed variables; FIGS. 3E and3F-illustrate deviations of the humanized or human origin antibodiesfrom the hypothetical optimal value of MNAC13 (calculated bothconsidering the degree of overall identity and of homology—in blue—andframework level—in magenta—); FIG. 3G depicts the structural alignmentwith the Fv fragment of MNAC13 of the respective regions of thehumanized or human origin antibodies, selected according to the degreeof identity and homology with the murine antibodies and to the degree ofresolution of available structural data; FIGS. 3H and 3I—depict thestructural alignment with the Fv fragment of MNAC13 (shown in cyan) ofthe respective region of the selected humanized antibody 1AD0 (shown inred) in 3H); and of the model of the antibodies resulting after CDRgrafting (shown in yellow at the framework level, in white at the CDRlevel) in 3I); FIG. 3J depicts a model of the Fv fragment of the MNAC13humanized antibody obtained as a result of the identification ofputative retro-mutations in the chosen framework (human origin residuesare shown in green and murine origin residues are shown in magenta).

FIGS. 4A through 4D depict distributions of the humanized or humanorigin antibodies (named using the PDB codes of their crystallographicstructures) according to the three analyzed variables; FIGS. 4E and 4Fdepict deviations of the humanized or human origin antibodies from thehypothetical optimal value of αD11 (calculated both considering thedegree of overall identity and of homology—in blue—and frameworklevel—in magenta—); FIG. 4G depict the structural alignment with the Fvfragment of αD11 of the respective regions of the humanized or humanorigin antibodies, selected according to the degree of identity andhomology with the murine antibodies and to the degree of resolution ofavailable structural data; FIGS. 4H and 4I depict the structuralalignment with the Fv fragment of αD11 (shown in cyan) of the respectiveregion of the selected humanized antibody 1JPS (shown in red) in 4H);and of the model of the antibodies resulting after CDR grafting (shownin yellow at the framework level, in white at the CDR level) in 4I);FIG. 4J depicts a model of the Fv fragment of the αD11 humanizedantibody obtained as a result of the identification of putativeretro-mutations in the chosen framework (human origin residues are shownin cyan and murine origin residues are shown in purple).

FIG. 5 depicts the alignment of the primary structures of the variableregions of the heavy chain (FIG. 5A) and of the light chain (FIG. 5B)respectively of MNAC13 (SEQ ID No. 22, SEQ ID No. 24), of the humanizedantibody selected for humanization (1AD0; SEQ ID No. 39, SEQ ID No. 40),of the humanized form of MNAC13 after CDR grafting on the framework of1AD0 and of the described retro-mutations and mutations (Hum MNAC13: SEQID No. 37, SEQ ID No. 38). CDRs are highlighted in the sequence of thehumanized form of the two chains of MNAC13 by underlined character.

FIG. 6 depicts the alignment of the primary structures of the variableregions of the heavy chain (FIG. 6A) and of the light chain (FIG. 6B)respectively of D11 (SEQ ID No. 2, SEQ ID No. 4), of the humanizedantibody selected for humanization (1JPS; SEQ ID No. 19, SEQ ID No. 20),of the humanized form of αD11 after CDR grafting on the framework of1AD0 and of the described retro-mutations and mutations (Hum αD11; SEQID No. 17, SEQ ID No. 18). CDRs are highlighted in the sequence of thehumanized form of the two chains of αD11 by underlined character.

FIG. 7A depicts the nucleotide sequence of the cDNA of the variableregion of the light chain of the murine form of MNAC13 (SEQ ID No. 23);FIG. 7B depicts the nucleotide sequence of the cDNA of the variableregion of the heavy chain of the murine form of MNAC13 (SEQ ID No. 21);FIGS. 7C and 7E depict the sequences of the oligonucleotides designed toobtain the humanized form of the variable region of the light chain ofMNAC13 (SEQ ID No. 38): L1S: SEQ ID No. 31; L2AS: SEQ ID No. 32; L3S:SEQ ID No. 33; L4AS: SEQ ID No. 34; L5S: SEQ ID No. 35; L6AS: SEQ ID No.36, by means of the overlap-assembly PCR technique, shown together withthe corresponding translation into amino acid sequence; FIGS. 7D and 7Fdepict the sequences of the oligonucleotides designed to obtain thehumanized form of the variable region of the heavy chain of MNAC13 (SEQID No. 37): H1S: SEQ ID No. 25; H2AS: SEQ ID No. 26; H3S: SEQ ID No. 27;H4AS: SEQ ID No. 28; H5S: SEQ ID No. 29; H6AS: SEQ ID No. 30, by meansof the overlap-assembly PCR technique, shown together with thecorresponding translation into amino acid sequence.

FIG. 8A depicts the nucleotide sequence of the cDNA of the variableregion of the light chain of the rat form of αD11 (SEQ ID No. 3); FIG.8B depicts the nucleotide sequence of the cDNA of the variable region ofthe heavy chain of the murine form of αD11 (SEQ ID No.1); FIGS. 8C and8E depict the sequences of the oligonucleotides designed to obtain thehumanized form of the variable region of the light chain of αD11 (SEQ IDNo. 18): L1S: SEQ ID No. 11; L2AS: SEQ ID No. 12; L3S: SEQ ID No. 13;L4AS: SEQ ID No. 14; L5S: SEQ ID No. 15; L6AS: SEQ ID No. 16, by meansof the overlap-assembly PCR technique, shown together with thecorresponding translation into amino acid sequence; FIGS. 8D and 8Fdepict the sequences of the oligonucleotides designed to obtain thehumanized form of the variable region of the heavy chain of D11 (SEQ IDNo. 17): H1S: SEQ ID No. 5; H2AS: SEQ ID No. 6; H3S: SEQ ID No. 7; H4AS:SEQ ID No. 8; H5S: SEQ ID No. 9; H6AS: SEQ ID No. 10, by means of theoverlap-assembly PCR technique, shown together with the correspondingtranslation into amino acid sequence.

FIG. 9 depicts maps of the plasmids used to clone the sequences of thehumanized variable regions of both antibodies obtained byoverlap-assembly PCR. FIG. 9A illustrates pVLexpress for the variabledomain of the light chain, FIG. 9B illustrates pVHexpress for thevariable domain of the heavy chain, FIG. 9C illustrates the plasmidresulting from cloning in pVLexpress the variable region of the lightchain of each humanized antibody, FIG. 9D illustrates alternativeconstructs obtained as a result of cloning in pVHexpress the variableregion of the heavy chain of each humanized antibody: 1) for theexpression in intact immunoglobulin form IgG1, 2) for expression in Fabfragment form, 3) for expression in immunotoxin form.

FIG. 10 depicts a comparison of the binding activity of the MNAC13antibody in chimeric form and in humanized form by means of ELISA assay,conducted immobilizing on plastic TrkA in immunoadhesin form: FIG. 10Adepicts a comparison between serial dilutions of supernatants oftransfected COS cells, subsequently concentrated; FIG. 10B depicts acomparison between serial dilutions of supernatants of transfected COScells purified by means of G sepharose protein.

FIG. 11 depicts the results of an assay of the binding activity of theαD11 antibody in humanized form by means of ELISA assay, conductedimmobilizing on plastic NGF.

DETAILED DESCRIPTION OF THE INVENTION

The method is based on the high resolution structural comparison for thehumanization of antibodies of in vivo therapeutic and diagnosticinterest. Moreover, humanized immunoglobulins are provided, able to bereactive specifically against the respective antigens (i.e. NGFneurotrophin and its TrkA receptor). Humanized immunoglobulins have aframework of human origin and they have one or more complementaritydetermining regions (CDRs) deriving from each original immunoglobulin(i.e. αD 11, a rat immunoglobulin, reactive specifically against NGF andMNAC13, a murine immunoglobulin, which specifically recognizes TrkA).Therefore, the immunoglobulins of the present invention, which may beeasily produced on a large scale, find therapeutic application not onlyin the therapy of NGF/TrkA dependent tumor forms, but also in thetreatment of chronic pain and inflammatory forms. Moreover, the specifichumanized immunoglobulin for the receptor has an additional diagnosticapplication for in vivo imaging both on TrkA positive tumors and oncells of the basal forebrain (as a precocious marker of Alzheimer'sdisease).

The present invention uses the recombinant segments of DNA coding theCDR regions of the light and/or heavy chain, able to bind an epitope ofinterest both on NGF and on TrkA, as in the case of the monoclonalantibodies αD11 and MNAC13 (respectively rat and mouse). The coding DNAsegments for these regions are joined to the DNA segments codingappropriate framework regions of human origin. The DNA sequences thatcode for the polypeptide chains comprising the CDRs of the light andheavy chain of the monoclonal antibodies MNAC13 and αD11 are included inFIGS. 7A, 7B and 8A, 8B respectively. Because of the degeneration of thegenetic code and of the substitutions of non critical amino acids, theDNA sequences can easily be modified. Moreover, DNA segments typicallyinclude an additional control sequence for the expression, operativelybound to the coding sequences for humanized immunoglobulins andcomprising regions of heterologous or naturally associated promoters.Preferably, the expression control sequences are systems with eukaryoticpromoters in vectors able to transform or transfect eukaryotic hostcells, but prokaryotic control sequences can be used as well. Once thevector is incorporated in the appropriate host, the host is maintainedin suitable conditions to assure a high level of expression. A furtherpurification follows of the light and heavy chains individually in theform of dimers, of intact antibodies or of other forms ofimmunoglobulins.

The sequences of coding DNA for the human constant region can beisolated by means of well known procedures from a variety of humancells, but preferably starting from immortalized B cells. The CDRs inthe immunoglobulins of the present invention are similarly derived fromthe monoclonal antibodies αD11 and MNAC13 able to bind respectively NGFand TrkA and products respectively in rat and mouse. Host cells suitablefor the expression and the secretion immunoglobulins can be obtainedfrom many sources such as the American Type Culture Collection(Catalogue of Cell Lines and Hybridomas, Fifth edition (1985) Rockville,Md., USA). Preferably, the CDRs incorporated in humanized antibodieshave sequences corresponding to those of the CDRs of αD11 and MNAC13 andcan include degenerated nucleotide sequences coding the correspondingamino acid sequences of the antibodies themselves.

Generally, the humanization design procedure is cyclical and iterativeand it comprises:

-   The analysis of the amino acid sequence of the murine antibody;-   The modeling of the corresponding Fv region;-   The analysis and selection of the amino acid sequence of the    acceptor framework of the human antibody;-   The identification of putative retro-mutations in the selected    framework;-   The design and the actual construction of the humanized antibody;-   The verification, by means of in vitro and/or in vivo assays, of the    maintained affinity and specificity of the binding.

If these activities are negatively influenced by the human framework, itwill be necessary to change the selection of the framework of theacceptor human antibodies, or to introduce compensating mutations.

Even if the choice of the human framework is configured as the mostcritical phase of the cycle, no general rules have been established todate. This depends on the fact that the advantages of the variouschoices (in terms of immunogenicity in the patient) have not beenaccurately studied from the clinical viewpoint. Therefore, to operatethe correct choice of the framework, only a series of approaches areavailable, which must be combined with the results obtained previously.

In particular, it is possible to use fixed frameworks (usually NEW forthe heavy chain and REI for the light chain, since their structures havebeen available for a long time). Another approach provides for the useof the frameworks found to be the most homologous in terms of sequencewith the antibody to be humanized. There are many databases to searchfor homologous human antibodies: the choice generally takes into accountthe length of the CDRs, the identity at the level of canonical residuesand of the residues at the interface level, in addition to a higherpercentage of identify between the sequences of the donor and of theacceptor. For a comparison between these two methods, see Graziano etal. (1995).

Moreover, according to a variant of the second approach the light chainand the heavy chain can be chosen from two different human antibodiescharacterized by a higher sequence homology. This approach was proposedby Riechmann et al. (1988) and by Shearman et al. (1991). In thisregard, in general, light and heavy chains deriving from the sameantibody have a higher probability of associating correctly, forming afunctional binding site, with respect to light and heavy chains derivingfrom different antibodies, although the fact that the interface betweenthe two chains is quite preserved can equally assure a correctinteraction. For a comparison between these two methods, see Roguska etal. (1996 and 1996)

Limiting the approach to a framework deriving from a particular humanantibody can entail the risk of incurring in somatic mutations whichproduce immunogenic epitopes even if the frameworks are of human origin.An alternative approach is to use frameworks based on human consensussequences, where idiosyncratic somatic mutations have been eliminated.The two approaches have been compared: in one case, no difference inbinding avidity was noted (Kolbinger et al., 1993), in another oneinstead the binding proved superior in the case of individual frameworks(Sato et al., 1994).

In any case, the consensus sequences themselves are artificial andtherefore, even if they have no idiosyncratic residues, they can createnon natural motives which are immunogenic. The alternative (Rosok etal., 1996) is to use germline human sequences collected in the V-BASEdatabase.

The non natural juxtaposition of the murine CDR regions with thevariable regions of the framework of human origin can give rise toconformational limits not represented in nature which, unless they arecorrected by the substitution of particular amino acid residues,determine the loss of binding affinity. The selection of the amino acidresidues to be substituted is partially determined by means of computermodeling. Hardware and software are available to producethree-dimensional images of immunoglobulin molecules. In general,molecular models are produced starting from already resolvedcrystallographic structures of immunoglobulin domains or chains. Thechains to be modeled are compared based on the amino acid resemblancewith chains or domains of resolved three-dimensional structures and thechains or the domains, which show the highest resemblance in terms ofsequence, are selected as starting points in the construction of themolecular model. However, the prediction of the antibody structure isnot always accurate. In particular, the third CDR region is difficult tomodel and it always represents a point of uncertainty in the structuralprediction of an antibody (Chothia et al., 1987). For this reason, as arule humanized antibodies, as a first approximation, have far lessbinding affinity and/or specificity towards the antigen than thestarting monoclonal antibody. This requires many successive cycles ofpoint mutations in the attempt to reconstitute the properties of thestarting antibody, with a trial and error procedure that cannot becompletely rationalized.

Considering the growing number of high resolution X-ray structures bothof available human and humanized antibodies, the intent was to avoid theuncertainties and ambiguities deriving from use of computer modeling,obtaining high resolution structural data for the Fab fragments of boththe antibodies of the invention by means of X-ray crystallography. Forthis purpose, both antibodies were purified from hybridoma, treatedproteolytically with papaine (a protease that cuts at the level of thejunction between CH1 and CH2 domain of the heavy chain) which givesorigin to the Fab fragments. As a result of the additional purification,both Fab fragments were crystallized and from two databases (low andhigh resolution), it was possible to solve the structures with theMolecular Substitution method and subsequently to refine them. Theapproach proposed by the invention, based on structural data obtainedexperimentally, provides a much more solid and rational starting point,both in the critical phase of the selection of the framework of theacceptor human antibody, and for the identification of putativeretro-mutations in the framework selected within the humanizationprocess of both neutralizing antibodies.

Amongst the various reported criteria which can guide the selection ofthe human antibody framework, the one used was the degree of identitybetween the antibody of murine and human origin at the primary sequence,to extend and complete its results with an analysis based on structuralalignment. A compared analysis of the corresponding structuresassociated to the original criterion assures a much more accuratecomparison and consequently a greater probability that the resultinghumanized antibody can preserve the characteristics of affinity andspecificity of the original murine antibody. Consequently, the strategyemployed combines the information deriving from the analysis andcomparison of amino acid sequences, both in terms of degree of identityand of level of homology, with the comparison of the respectivethree-dimensional structures.

In particular, the information deriving from the optimal alignment ofthe primary structures has a dual role. In the first place, thisanalysis allows to reduce the number of possible tertiary structures tobe compared, limiting itself to those characterized by a high degree ofhomology and identity. Among these sequences characterized by an optimalalignment at the primary structure level and for which structural dataare available, a further selection was conducted, concentrating only onthe resolved structures with high resolution or otherwise withresolution comparable to that of the structures obtained by us (i.e. nogreater than 2.5 Å). This approach assures a much more accuratealignment of the tertiary structures and much more significant estimatesof the structural differences, expressed in RMS (root mean squaredeviation: square root of the mean square deviation; Carugo and Pongor,2001 and 2003). Low resolution data provide rather indicative, anddefinitely less precise information on the actual relative position ofeach individual atom in space.

To assess the degree of superposition of each individual structure, ofhuman origin or engineered, the RMS was calculated between atoms ofalpha carbon constituting the respective amino acid skeletons, notconsidering atom pairs with an RMS exceeding 2 Å. From this analysis, aninformation is obtained which must therefore take into account not onlythe diversity between the structures (expressed by the value of RMS),but also the percentage of atoms of alpha carbon actually employed incalculating each RMS.

These tertiary structure level resemblance data were associated to thecomparative analysis of the primary sequences both in terms of identityand of homology.

It is hence deduced that the selection of the optimal framework forhumanization is configured as a three-variable problem, which can thusbe represented in space, both when associating the homology level andthe degree of identity to the structural alignment. This type ofanalysis was then conducted also reducing the regions in question in thetwo types of alignment to the regions of the respective frameworks.Comparing the distributions of the antibodies considered in the space ofthe three analyzed variables (respectively, value of RMS, percentages ofatoms on which RMS was calculated and a similitude index between primarystructures, i.e. percentage of overall identity, of overall homology, ofidentity at the framework level, of homology at the framework level)with the optimal position in the space of the three variables that eachantibody would occupy if it were of human origin, it is possibly clearlyto identify the human origin antibody that most approaches this idealposition at the level of primary and tertiary structure. To rationalizethis result, in each of the four analyses the deviations from thehypothetical optimal position are calculated for each position of thehumanized or human origin antibodies considered.

On the basis of this method of selection, it is possible to choose theacceptor framework in the subsequent process of CDR grafting for thehumanization of a given antibody. In general, it is necessary tominimize the substitutions of amino acid residues of human origin withresidues of murine origin, for the introduction of murine residuesincreases the risk that the antibody will induce a HAMA response in thehuman patient. On the other hand, the complementarity determiningregions (CDRs) contain the residues with the greater probability ofinteracting with the antigen and for this reason they must be maintainedin the humanized antibody. They are defined by means of the sequenceaccording to Kabat or by means of the structure according to Chothia.The advantage of using the second system to define them is that ingeneral the CDRs are shorter and hence the humanized antibody isconstituted by a lesser fraction of xenogenic fragments. In any case ithas been demonstrated that generally following Kabat's definitions it ispossible drastically to reduce the number of cycles required forhumanization. Once the CDRs are defined, it is necessary to identify thecanonical classes (defined by Chothia and Lesk) to which they belong andsubsequently maintain the canonical residues in the humanizedantibodies.

It is also essential to analyze the residues that mediate theinteraction between the light chain and the heavy chain of the variabledomains (Table 1), maintaining any unusual residues in the humanizedantibody (Singer et al., 1993; Daugherty et al.; 1991; De Martino etal., 1991).

Moreover, further amino acids to be maintained are selected based ontheir possible influence on the conformation of the CDRs and/or on theinteraction with the antigen. When the amino acid differs between theframework of animal origin and the equivalent acceptor framework ofhuman original, the amino acid of the acceptor framework should besubstituted by the equivalent murine residue, if it is reasonable toexpect that the amino acid is in direct non covalent contact with theantigen, or is adjacent to a CDR region, or in any case interacts with aCDR region (it is situated within 4-6 Å from a CDR region).

TABLE 1 Residues that mediate the interaction between the light chainand the heavy chain of the variable domains LIGHT VARIABLE CHAIN L HEAVYVARIABLE CHAIN H Kabat Kabat Position Mouse Human Position Mouse Human34 H678 N420 A531 N147 35 H1001 N636 S527 H340 A408 Y147 D66 S402 E184G167 A143 E114 36 Y1653 F198 Y748 F80 37 V2336 I200 V1037 I477 L96 L2738 Q1865 H47 Q799 H22 39 Q2518 K67 Q1539 R16 44 (A) P1767 V132 P839 L545 (A) L2636 P16 L1531 P24 I40 46 L1381 R374 L760 V37 47 W2518 L64 W1534Y21 P97 Y50 87 Y1457 F448 Y795 F41 91 Y2149 F479 Y1429 F116 89 Q1170L206 Q687 M107 93 A2202 T222 A1346 T90 F144 V102 V71 91 W376 S374 Y404R115 95 Y399 G375 D268 G266 G356 Y295 S105 A84 S340 D340 R109 E100 H182R226 96 (A) L537 Y380 L134 Y215 100k (A)  F1285 M450 F540 M109 W285 F78W73 I71 L33 98 (A) F1724 F654 103 (A)  W1469 W323

In particular, a further analysis involves other residues which definethe so-called Vernier zone, a zone that stabilizes the structure of theCDRs; it is important to maintain the characteristics of this region.

Other residues which are candidates for mutation are amino acids of theacceptor framework which are unusual for a human immunoglobulin in thatposition. These residues can be substituted with amino acids derivingfrom the equivalent position of more typical human immunoglobulins oralternatively residues originating from the equivalent position of thedonor framework can be introduced into the acceptor framework when saidamino acids are typical for the human immunoglobulins in thoseparticular positions.

Moreover, again on the basis of the consensus sequences of humanimmunoglobulins, mutations are introduced in the humanized form whichinsert residues preserved in the human instead of the unusual residuespresent both in the donor and in the acceptor framework.

The respective pairs of crystallographic structures are then modified,first effecting the grafting of the CDRs of animal origin in the humanframeworks. Then, all the mutations and retro-mutations described aboveare introduced. The modified structures are then assembled in compositeimmunoglobulins. The resulting models are refined by minimizingmechanical energy (in terms of torsion angles and binding angles anddistances) using the force field.

For all other regions, different from the specific amino acidsubstitutions discussed above, the framework regions of the humanizedimmunoglobulins are usually substantially identical to the frameworkregions of the human antibodies from which they were derived. In anycase in these engineered proteins obtained by grafting, the frameworkregions can vary relative to the native sequence at the primarystructure level due to many amino acid substitutions, deletions orinsertions, terminal or intermediate, and other changes. Naturally, mostof the residues in the framework region brings a very small or evennon-existent contribution to the specificity or affinity of an antibody.Therefore, many individual conservative substitutions in the residues ofthe framework can be tolerated without appreciable variations of thespecificity or affinity in the resulting humanized immunoglobulin. Ingeneral, nevertheless, such substitutions are not desirable. It ispossible to obtain modifications in the nucleotide sequence with avariety of widely employed techniques, such as site-specific mutagenesis(Gillman & Smith, 1979; Roberts et al., 1987).

Alternative, polypeptide fragments can be produced, comprising only apart of the primary structure of the antibody, which fragments retainone or more peculiar activities of the immunoglobulins (e.g., thebinding activity). These polypeptide fragments can be produced by meansof proteolytic digestion starting from intact antibodies or insertingstop codons in the desired positions in the carriers bearing the codingDNA sequences for the variable regions of the heavy and light chain bymeans of site specific mutagenesis (in particular after the CH1 regionto produce Fab fragments or after the hinge region to produce (Fab′)₂fragments. Antibodies in the form of scFv can be obtained by joining thevariable regions of the heavy chain and of the light chain by means of alinker (Huston et al., 1988; Bird et al., 1988). The Fv or Fab fragmentscan be expressed in E. coli (Buchner and Rudolph, 1991; Skerra et al.,1991) or also in eukaryotic cells, preferably mammal derived.Considering that like many other genes, the genes of the immunoglobulinsuper family contain distinct functional regions, each characterized byone or more specific biological activities, the genes can be fused tofunctional regions deriving from other genes (e.g. enzymes) to producefusion proteins (e.g. immunotoxins) provided with new properties.

The expression of humanized immunoglobulin sequences in bacteria can beused to select humanized immunoglobulin sequences, characterized byhigher affinity mutagenizing the CDR regions and producing phagelibraries for phage display. Using these libraries, it is possible toperform a screening in the search for variants at the level of the CDRsof the humanized immunoglobulins that have a higher affinity and/orbinding specificity for the antigens. Methods to obtain phage-displaylibraries bearing sequences of the variable regions of immunoglobulinshave been amply reported (Cesareni, 1992; Swimmer et al., 1992; Gram etal, 1992; Clackson et al., 1991; Scott & Smith, 1990; Garrard et al.,1991). The sequences resulting from the variants of humanizedimmunoglobulins, whose CDRs were thus remodeled, are subsequentlyexpressed in a host that is suitable to assure a high expressionthereof.

As stated above, the DNA sequences are expressed in the host cells afterbeing operatively bound (i.e. positioned in such a way as to assuretheir functionality) to expression control sequences. These carriers cantypically be replicated in the host organism as episomes or as anintegral part of the chromosome DNA. Commonly, the expression carrierscontain a selectable marker to allow one to identify the cells that havebeen transformed with the DNA sequences of interest.

For the production of the humanized immunoglobulins of the invention inrecombinant form of scFv or in Fab form, prokaryotic systems arepreferred. E. coli is one of the prokaryotic hosts that is particularlyuseful for cloning the DNA sequences of the present invention. Moreover,a great number of well characterized promoters is available, e.g. lac ortrp operon or β-lactamase or λ phage. Typically, these promoters controlexpression and bear binding site for the ribosome, for the correct startand finish of transcription and translation. It is possible to increasethe half-life of the humanized immunoglobulins of the invention producedin prokaryotic systems by conjugation with polyethylene glycol (PEG).

Other single-cell organisms, such as yeasts, can be used for expression.The host of choice is Saccharomyces, using suitable carriers providedwith expression control, replication termination and origin sequences.

Insect cell cultures can also be used to produce the humanizedimmunoglobulins of the invention, typically using cells of S2 Drosophilatransfected in stable fashion or cells of Spodoptera frugiperda with theexpression system based on the Baculovirus (Putlitz et al., 1990).

Plants and cultures of vegetable cells can be used for the expression ofthe humanized immunoglobulins of the invention. (Larrick & Fry, 1991;Benvenuto et al., 1991; Durin et al., 1990; Hiatt et al, 1989).

However, in all these cases it is impossible to obtain the correct typeof glycosylation necessary to assure the effector function in theactivation of the human immune system. For this purpose, it is possibleto use tissue cultures of mammal cells to express the polypeptides ofthe present invention in integral form of IgG1, which have proven to bethe most effective isotype among seric immunoglobulins in the inductionof the immune response (Winnacker, 1987). It should be stressed that,considering that the isotype determines the lytic potential of anantibody, generally the IgG1 isotype is used for therapeutic purposes(since it induces the immune response, both cell-mediated and mediatedby the system of the complement), whilst the IgG4 is used for diagnosticapplications (Riechmann et al., 1988). In particular, mammal cell arepreferred, considering the great number of host cell lines developed forthe secretion of intact immunoglobulins, among them the CHO cell lines,several lines of COS, the HeLa cells, myeloma cell lines (NS0, SP/2,YB/0 e P3X63.Ag8.653), transformed B cells or hybridomas. Expressioncarriers for these cells can include expression control sequences, suchas a replication origin, a promoter, an enhancer (Queen et al., 1986),and the sequences required for ribosome binding, RNA splicing andpolyadenylation, and sequences for transcription termination. Theexpression control sequences of choice are promoters deriving fromimmunoglobulin genes and from viruses, such as SV40, Adenovirus, BovinePapilloma Virus, Cytomegalovirus and the like. Generally, the expressionvector includes a selectable marker, such as the resistance to neomycin.For the expression of humanized antibodies, it is preferable tocultivate the mammal cell lines with a serum-free medium. For example,the HUDREG-55 cell line can easily be grown in Serum-Free andProtein-Free Hybridoma Medium Cat. No. S-2897 from Sigma (St. Louis,Mo.).

The genes coding the humanized immunoglobulins of the invention can beused to generate non human transgenic animals, which express thehumanized immunoglobulins of interest, typically in a retrievable bodyfluid such as milk or serum. Such transgenes comprise the polynucleotidesequence coding the humanized immunoglobulins operatively bound to apromoter, usually with an enhancer sequence, such as that of the rodentimmunoglobulin or the promoter/enhancer of the casein gene (Buhler etal., 1990; Meade et al, 1990). The transgenes can be transferred intothe cells or embryos by means of homologous recombination constructs.Among non human animals used: mouse, rat, sheep, bovine and goat(WO91/08216).

Once they are expressed as intact antibodies, their dimers, theindividual light and heavy chains, or in other forms the immunoglobulinsof the present invention can be purified following standard procedures,such as precipitation with ammonium sulfate, affinity columns,chromatography on column (Scopes, 1982). For pharmaceuticalapplications, substantially pure immunoglobulins are necessary, withminimum homogeneity between 90 and 95%, but preferably between 98 and99% or even higher. Once purified, partially or to the desiredhomogeneity, proteins can be used for therapeutic use (also inextra-body fashion), for diagnostic use (imaging for the diagnostics oftumors or of Alzheimer's Disease) or to develop and perform biochemicalassays, immunofluorescent colorings and the like (see, in general,Lefkovits and Pernis, 1979 and 1981).

A pharmaceutical application of the present invention pertains to theuse of humanized immunoglobulin MNAC13 in the form of immunotoxin toeliminate TrkA-expressing cells (in the case of pancreas and prostatetumors). The immunotoxins are characterized by two components and areparticularly suitable to kill particular cells both in vitro and invivo. One component of the cytotoxic agent that is generally lethal fora cell is absorbed or if it interacts with the cell surface. The secondcomponent provides the means to address the toxic agent to a specifictarget cell type, such as the cells that express the epitope of the TrkAreceptor. The two components are chemically bound to each other by meansof any one of the great variety of chemical procedures available. Forexample, when the cytotoxic agent is a protein and the second componentis an intact immunoglobulin the link can be mediated by cross-bindingand heterobifunctional agents (SPDP, carbodiimide, glutaraldehyde).Alternatively, the two components can be bound genetically (Chaudhary etal., 1989). The production of various immunotoxins is reported by Thorpeet al. (1982).

A great number of cytotoxic agents are suitable for application asimmunotoxins. Cytotoxic agents can include radionuclides such as iodine131 or other isotopes of iodine, yttrium 90, rhenium 188 and bismuth 212or other isotopes that emit alpha particles, a great number ofchemotherapeutic drugs such as vindesin, methotrexate, adriamycin andcisplatin; and cytotoxic proteins, such as proteins that inhibit andribosomes (such as the pokeweed antiviral protein, the Pseudomonasexotoxin A and the diphteric toxin, ricin A and clavin of vegetableorigin) or agents active at the cell surface level (such asphospholipase enzymes such as phospholipase C)—eds. Baldwin and Byers,1985; U.S. Ser. No. 07/290,968; Olsnes and Phil, 1982. It should bestressed that the cytoxic region of the immunotoxin can itself beimmunogenic and consequently limit the clinical usefulness of the fusionprotein in case of chronic or long term therapy. An alternative to avoidthe problem of the immunogenicity of the toxin is to express in fusionwith the binding domain of the antibody a protein able to interact withthe DNA and bind to this fusion protein the expression carrier thatcontains the toxin expression cassette. The numerous positive charges ofprotamin, a human protein that binds the DNA, can interact in stablefashion with the negative charges of the DNA, generating a fusionpartner for the neutral charge antibody, much more stable and lessimmunogenic than the toxin itself. After internalization of theantibody-plasmid complex via receptor mediated endocytosis, theexpression of the toxin causes the death of the cell. Moreover,selectivity towards the target cell to be eliminated can be furtherenhanced by inserting inducible or cell-specific promoters into thetoxin expression cassette. This approach is aimed at maximizing theselective elimination of tumor cells while minimizing toxicity sideeffects (Chen et al., 1995).

The component that addresses the immunotoxin to the correct targetincludes the MNAC13 humanized immunoglobulin of the present invention inthe form of intact immunoglobulin or of the binding fragment or as Fabor Fv fragment. Typically, the antibodies in the immunotoxins are of thehuman isotype IgM or IgG, but other constant regions can be used aswell.

The antibodies and the pharmaceutical compositions of this invention areparticularly useful for administration, following any effectivemethodology to address the antibodies at the level of the tissueinvolved in the pathology. This includes (but is not limited to):intraperitoneal, intramuscular, intravenous, subcutaneous,intratracheal, oral, enteral, parenteral, intranasal or dermaladministration. The antibodies of the present invention can typically beadministered for local application by injection (intraperitoneal orintracranial—typically in a cerebral ventricle—or intrapericardiac orintrabursal) of liquid formulations or by ingestion of solidformulations (in the form of pills, tablets, capsules) or of liquidformulations (in the form of emulsions and solutions). Compositions forparenteral administration commonly comprise a solution of immunoglobulindissolved in a compatible, preferably aqueous solution. Theconcentration of the antibody in these formulations can vary from lessthan 0.005% to 15-20% and it is selected mainly according to the volumesof the liquid, its viscosity, etc., and according to the particularadministration mode selected.

Alternatively, the antibodies can be prepared for administration insolid form. The antibodies can be combined with different inert orexcipient substances, which can include ligands such as microcrystallinecellulose, gelatin or Arabic rubber; recipients such lactose or starch;agents such as alginic acid, Primogel or corn starch; lubricants such asmagnesium stearate, colloidal silicon dioxide; sweeteners such assaccharose or saccharin; or flavors, such as mint and methyl salicylate.Other pharmaceutical administration systems include hydrogel,hydroxymethylcellulose, liposomes, microcapsules, microemulsions,microspheres, etc. Local injections directly in the tissues affected byillness such as tumors is a preferential method for the administrationof the antibodies of the present invention.

The antibodies of the invention can be frozen or lyophilized andreconstituted immediately before use in a suitable buffer. Consideringthat lyophilization and reconstitution can determine a variable loss inthe activity of the antibody (for conventional immunoglobulins, classIgM antibodies tend to have a greater loss of activities than class IgGantibodies), administration levels must be calibrated to compensate forthis fact.

Thanks to their high blocking capacity, the compositions containing theantibodies of the present invention can be administered for prophylacticand/or therapeutic treatments to prevent or reduce the inflammatorycomponent associated to pathological situations or chronic pain, inparticular chronic visceral pain (associated to physiological disorders,such as dysmenorrhea, dyspepsia, gastrointestinal reflux, pancreatitis,visceralgia or irritable intestine syndrome).

In prophylactic applications, compositions containing antibodies of thepresent invention are administered to patients who do not yet sufferfrom a particular pathology to enhance their resistance.

The antibodies of the present invention also provide a method forreducing the volume of prostate or pancreas tumors and for preventingfurther tumor growth or reduce the rate of growth of the tumor. Thiseffect can be mediated by both the humanized antibodies of the presentinvention because they are extremely effective in the neutralization ofthe interaction between NGF and TrkA, necessary to sustain tumor growthand progression in autocrine or paracrine fashion. Moreover thehumanized form of MNAC13 interacts with a membrane receptor and hencecan also be used for the direct elimination of neoplastic cells becausethey are able to activate the host's immune response (if administered inthe form of IgG1) or to convey a cytotoxic agent localizing it at thelevel of the cancerous mass (if administered in the form ofimmunotoxin). Their administration in the tumor site preferably takesplace through direct and localized injection into the tissue or near thetumor site. For systemic administration, doses vary from 0.05 mg/kg perday to 500 mg/kg per day, although dosages in the lower region of therange are preferred because they are easier to administer. Dosages canbe calibrated for example to guarantee a particular level in the plasmaof the antibody (in the range of about 5-30 mg/ml, preferably between10-15 mg/ml) and maintain this level for a given period of time untilthe clinical results are achieved. Humanized antibodies should beeliminated much more slowly and require lower dosages to maintain aneffective level in the plasma; moreover, considering the high affinity,administration is less frequent and less sizable than with antibodieshaving lower affinity. The therapeutically effective dosage of eachantibody can be determined during the treatment, based on the reductionin the volume of the tumor or on the rate of growth of the tumor orideally on the total disappearance of the cancerous pathological state.Effective methods for measuring or assessing the stage of pancreatic orprostatic tumors are based on the measurement of the prostate specificantigen (PSA) in blood, on the measurement of the survival time forpancreas tumors, on the measurement of the slowing or inhibition ofdiffusion for metastases in the case of both tumor.

For direct injection at the level of the tumor site, dosage depends ondifferent factors including the type, stage and volume of the tumor,along with many other variables. Depending on tumor volume, typicaltherapeutic doses may vary from 0.01 mg/mm and 10 mg/mm injections whichcan be administered with the necessary frequency. Another method toassess the effectiveness of a particular treatment is to evaluate theinhibition of the TrkA receptor, e.g. by measuring its activity by meansof ELISA assay (Angeles et al., 1996).

It is important to stress that, TrkA is configured not only as atherapeutic target but also as a diagnostic target for in vivo imaging,e.g. for imaging of TrkA positive tumors (as a positive or negativemarker, depending on tumor type and origin) and imaging on cells of thebasal forebrain (as a precocious marker of insurgence of Alzheimer'sdisease). The MNAC13 humanized antibody of the present invention canalso find a wide variety of in vitro applications (ELISA, IRMA, RIA,immunohistochemistry).

For diagnostic purposes, the antibodies can be both marked and unmarked.Unmarked antibodies can be used in combination with other markedantibodies (secondary antibodies), which are reactive against humanized,or human antibodies (e.g. specific antibodies for the constant regionsof human immunoglobulins). Alternatively, antibodies can be markeddirectly. A wide variety of markings can be used, e.g. radionuclides,fluorophores, colorings, enzymes, enzymatic substrates, enzymaticfactors, enzymatic inhibitors, ligands (in particular aptenic), etc.Numerous types of immunologic assays are available in the sector.

In particular, for imaging diagnostic applications, to the antibody isconjugated an agent that is detectable or marked in isotopic manner(using radioisotopes of iodine, indium, technetium) or in paramagneticmanner (paramagnetic atoms or ions, such as transition elements,actinides and rare earths; in particular, manganese II, copper II andcobalt II) as described by Goding (1986) and Paik et al. (1982). Imagingprocedures entail the intravenous, intraperitoneal or subcutaneousinjection (in lymphatic drainage regions to identify lymph nodemetastases) and they use detectors of radionuclide emissions (such asscintillation .beta. counters) in the case of immunoscintigraphy; if aparamagnetic marking is used instead, an NMR (Nuclear MagneticResonance) spectrometer is used. The invention shall now be described inits non limiting embodiments, with reference to the following figures:

Results X-Ray Structures of the Fab Fragment of the MNAC13 and αD11Monoclonal Antibodies

Both monoclonal antibodies were obtained and purified according tostandard procedures. The MNAC13 IgG1 and αD11 IgG2a immunoglobulins wereexpressed in the supernatant by means of culture of hybridoma cells andconcentrated by precipitation with 29% ammonium sulfate followed bydialysis in PBS. Both immunoglobulins were purified by affinitychromatography using a column of Protein G Sepharose (Pharmacia).

Following dialysis in phosphate buffer 10 mM pH 7, EDTA 20 mM usingSpectra-Por 12/14K membranes (Spectrum) at 4° C., each sample wasconcentrated by means of Centricon 50 KDa ultrafiltration units (Amicon)and incubated with 13 mM Cys and treated with immobilized papaine(Pierce) (with an enzyme:substrate ratio of 1:15) for 5 h at 37° C. Theprocedure for purifying the respective Fab fragments is diversified,although it is always based on ionic exchange chromatography.

In the case of MNAC13, after dialysis against Tris HCl 100 mM pH 8.0, itwas possible to eliminate the Fc fragments through a DEAE-Sephacelcolumn (Pharmacia) balanced with the same buffer. FabMNAC13 wascollected in the excluded volume whilst the Fc fragments and a fractionof undigested IgG1 were eluted with 250 mM NaCl. The Fab fragment wasseparated from undigested IgG1 by gel filtration on a Superdex G75(Pharmacia) column balanced with Tris HCl 100 mM pH 8.0, NaCl 150 mM.The homogeneity and purity of the fractions was controlled byelectrophoretic separation on 12% polyacrylamide gel followed bycoloring with Coomassie (FIG. 1A). The concentration of the purifiedprotein was determined by means of Lowry assay (Bio-Rad). From 11 ofhybridoma surnatant, it was possible to obtain up to 3 mg of MNAC13 Fab(with purity exceeding 99%).

In terms of the purification of the Fab fragment of the αD11 antibody,the sample treated with papaine was dialyzed against 10 mM pH 7.8phosphate buffer: the Fc fragments were eliminated through aDEAE-Sephacel column (Pharmacia) balanced with this same buffer. The Fabfragment of αD11 was collected in the excluded volume, whilst the Fcfragments and a fraction of undigested IgG2a were eluted with 250 mM pH6.8 phosphate buffer. The Fab fragment was separated from the undigestedIgG2a by means of filtration gel on a Superdex G75 column (Pharmacia)balanced with 10 mM pH 7.8 phosphate buffer, NaCl 150 mM. Thehomogeneity and purity of the fractions was controlled byelectrophoretic separation on 12% polyacrylamide gel followed bycoloring with Coomassie (FIG. 2A). The concentration of the purifiedprotein was determined by means of Lowry assay (Bio-Rad). From 11 ofhybridoma surnatant, it was possible to obtain up to 6 mg of αD11 Fab(with purity exceeding 99%). Both the Fab fragment of the MNAC13antibody purified in 10 mM Tris pH 8.0, 50 mM NaCl, and the Fab fragmentof the αD11 antibody purified in 10 mM Na phosphate pH 7.8 and 50 mMNaCl were concentrated to 5-10 mg/ml by means of Centricon 30 KDaultrafiltration unit (Amicon). The crystallization experiments wereconducted following the hanging-drop method at 16.degree.C. following afactorial combination approach (Jancarik & Kim, 1991) using CrystalScreen I and II (Hampton Research-Laguna Niguel, Calif., USA-) andScreening Kit (Jena BioSciences).

Drops of 2 μl of the concentrated protein sample were added to an equalvolume of the solution containing the precipitant agent and balanced bydiffusion with a solution in the reservoir (0.7 ml) in 24 well Linbroplates.

In terms of the Fab fragment of the MNAC13 antibody, the most promisinginitial result, obtained with equal volumes of protein and precipitantcontaining 2M ammonium sulfate, 5% v/v isopropanol (Crystal Screen II,Reactant #5), was optimized until obtaining crystals that grow in aboutone week, similar to what is shown in FIG. 1B.

In terms of the Fab fragment of the αD11 antibody, the most promisinginitial result, obtained using equal volumes of protein and precipitantcontaining 20% PEG4000, 0.6M NaCl, 100 mM MES pH 6.5 (Kit number 4,solution C2), required a long optimization process, modifying thecomposition of the precipitant agent to PEG4000, 0.6M NaCl, 100 mM BTPpH 5.5 and the ratios between protein and precipitant solution (1.5:1)until obtaining crystals that grow in about one week, similar to what isshown in FIG. 2B.

In both cases, an initial set of low resolution data was collected onthe XRD1 diffraction line of the ELETTRA synchrotron (Trieste, Italy),and then a second, more complete set of data at higher resolution wascollected on the ID14-EH1 diffraction line of the ESRF synchrotron(Grenoble, France) The crystals were frozen under liquid nitrogen flowwith the cooling system by Oxford Cryosystems (Oxford, UK), using in thecase of the Fab fragment of the MNAC13 antibody a solution containing2.2 M ammonium sulfate, 6% v/v isopropanol and 20% v/v glycerol ascryoprotector. A representative high resolution diffraction spectrum foreach protein is shown in FIGS. 1B and 2B. All four sets of X-raydiffraction data were processed, indexed, integrated and subsequentlyscaled using the DENZO and SCALEPACK programs (Otwinowski & Minor, 1997)respectively, while the CCP4 (Collaborative Computational Project,Number 4, 1994) package was used for data reduction. The statistics forthe collection and processing of high and low resolution data of thecrystals of the Fab fragment of the MNAC13 antibody are set out in thefollowing table:

X-ray source ELETTRA ESRF Wavelength (Å) 1.000 0.934 Detector mar345marCCD Spatial group P2₁2₁2₁ P2₁2₁2₁ Parameters of the unitary cell a(Å) 52.78 52.73 b (Å) 67.53 67.55 c (Å) 111.51 111.43 Mosaicity (°) 0.400.47 Resolution interval (Å) 12.0-2.50 17.0-1.80 (2.59-2.50) (1.83-1.80)No. measures 98688 414115 No. of reflexes observed with 56918 227914 I ≧0 No. of unique reflexes with 14203 (1371) 38392 (1893) I ≧ 0Completeness (%) 99.5 (99.3) 99.5 (99.6) Redundancy 4.0 (4.0) 5.9 (4.9)<I/σ (I)> of the measured data 9.4 (4.7) 8.2 (1.1) R_(sym) (%) 5.7(15.2) 6.3 (39.8)

Similarly, the following table summarizes the statistics for thecollection and processing of high and low resolution data of thecrystals of the Fab fragment of the αD11 antibody:

X-ray source ELETTRA ESRF Wavelength (Å) 1.000 0.934 Detector marCCDmarCCD Spatial group P1 C2 Parameters of the unitary cell a (Å) 42.685114.801 b (Å) 50.626 69.354 c (Å) 102.697 64.104 α (°) 81.977 90 β(°)89.116 117.02 γ(°) 85.957 90 Mosaicity (°) 0.44 0.40 Resolution interval(Å) 47.6-2.57 17.0-1.70 (2.8-2.7) (1.75-1.70) No. measures 124456 492594No. of reflexes observed with 74241 399184 I ≧ 0 No. of unique reflexeswith 23413 (2162) 47951 (3198) I ≧ 0 Completeness (%) 98.2 (92.4) 97.2(78.4) Redundancy 5.7 (5.2) 6.7 (7.5) <I/σ (I)> of the measured data29.6 (6.7) 9.5 (2.1) R_(sym) (%) 11.0 (33.5) 5.8 (27.8)

The values in parenthesis refer to the shell with the highestresolution. Considering the high number of available structures of Fabfragments, the most convenient method to determine the structure of bothproteins was Molecular Substitution. In a research in the Protein DataBank (Berman et al., 2000) for homologous structures, the selectioncriteria gave priority to the combination between comparable resolutionand highest level of sequence identity. On these bases, respectively,were selected for MNAC13: 1BM3: the structure of the complex between Fabfragment of the Opg2 Fab immunoglobulin and the peptide recognized by it(Kodandapani et al., 1999), resolved at a resolution 2.0 Å and providedwith a sequence identity respectively of 70 and 88% for the heavy andlight chain,

for αD11: 1CIC: the structure of the complex of idiotype-anti-idiotypeFab fragments FabD1.3-FabE225 (Bentley et al., 1990), resolved at aresolution 2.5 Å and provided with a sequence identity respectively of82 and 82.65% for the heavy and light chain. The determination of bothstructures was obtained by the Molecular Substitution method using theAMoRe program (Navaza, 1994), with the respective models usingseparately the constant domains and variable domains considering theextreme variability of the angle formed by the axis of binarypseudosymmetry between the variable and constant regions. The solutionobtained in the determination of the structure, of the Fab fragment ofMNAC13 following refined with rigid body is shown in the followingtable:

Peak α β γ x y z C_(f) R_(f) C_(I) C_(p) V 106.5 20.7 143.9 .1004 .0757.04680 C 94.5 13.9 173.3 .1684 .3073 .7355 53.7 39.8 54.8 32.4

Similarly, the solution obtained in the determination of the structureof the Fab fragment of αD11 following refinement with rigid body for thespatial group C2 is shown in the following table:

Peak α β γ x y z C_(f) R_(f) C_(I) C_(p) V 151.0 155.4 43.0 .1424 .0005.449 C 17.8 63.7 73.2 .3625 .9532 .1991 55.0 38.9 49.7 35.9 Where V =variable domain C = constant domain α, β, γ = Eurelian angles (°). x, y,z = Translation (fractionary). C_(f) = Correlation of the amplitudes(×100) R_(f) = Crystallographic R factor. C_(I) = Correlation of theintensities (×100). C_(p) = Correlation of the truncated Pattersonfunction (×100).

The subsequent refinement of the two structures was obtained by means ofa cyclic procedures, comprising two alternated phase: manualconstruction of the model using the interactive software for computergraphics “O” (Kleywegt and Jones, 1994); positional refinement andrefinement of B isotropic thermal factors using automatic protocols ofthe CNS suite, Crystallography and NMR System (Brunger et al., 1998).The procedure after some phases of refinement with rigid body,contemplated different refinement cycles. Once the insertion of allmutations and deletion is completed to complete the models, thelocalization of the water molecules and any ions and ligands wasconducted. At the end, maintaining the model as close as possible to theideal values in terms of stereochemical, the positional weight wa andthe weight of the thermal factor B r-weight were optimized. Thestatistics and the final parameters that describe the quality of themodel obtained for the Fab fragment of the MNAC13 antibody aresummarized in the following table:

Number of protein atoms 3244 Number of solvent atoms 351 Number ofsulfate ions 4 Number of Tris molecules 1 Number of isopropanolmolecules 1 Resolution interval (Å) 39-1.778 Final R factor 19.35% FinalR_(free) factor (calculated on 10% of the data) 23.22% Rms DeviationsBinding distances (Å) 0.008 Binding angles (°) 1.456 Dihedral angles (°)27.29 Improper angles (°) 0.928 Mean Isotrope Thermal Factor (A²)Complete protein 23.55 Light chain 24.14 Heavy chain 22.99 Watermolecules 31.95 Ions (sulfate) 55.94 Tris 46.06 Isopropanol 32.60

Similarly, the statistics and the final parameters that describe thequality of the model obtained for the Fab fragment of the αD11 antibodyare summarized in the following table:

Number of protein atoms 3229 Number of solvent atoms 403 Number ofchloride ions 1 Resolution interval (Å) 30-1.70 Final R factor 19.54%Final R_(free) factor (calculated on 10% of the data) 24.22% RmsDeviations Binding distances (Å) 0.0096 Angoli di legame (°) 1.6571Binding angles (°) 1.6571 Angoli dieDihedral angle (°) 27.40 Improperangles (°) 1.048 Mean Isotrope Thermal Factor (A²) Complete protein25.58 Light chain 24.14 Heavy chain 22.99 Water molecules 38.80 Ions(chloride) 20.58

Moreover, both models were examined by final geometry analysis with thePROCHECK suite (Laskowski et al., 1993) as shown in the respectivetables and in the respective Ramachandran charts (FIGS. 1D and 2D).

Use of the X-Ray Structures of the Fab Fragment of the MNAC13 and αD11Monoclonal Antibodies in the Selection of a Framework of Human Origin

In the selection of human antibody framework, the approach describedabove was follows, which combines on the degree of identity between theantibody of murine and human origin at the primary sequence level to thedegree of structural similarity of the polypeptide skeletons.

In particular, a series of possible acceptor frameworks of human originor humanized antibodies was selected on the basis of the highest levelboth of homology and of identity of the primary structures by a searchin the BLAST database. This selection was conducted for both blockingantibodies both considering the entire variable regions of theantibodies and narrowing the search to the framework regions.

Within each group of selected antibodies, only those for whichstructural data with high resolution or otherwise with resolutioncomparable to that of the structures obtained by us (i.e. no greaterthan 2.5 Å are available were considered, conducting a search in PDB(Protein Data Bank). The respective amino acid skeleton were thensuperimposed using the “superimpose” software (Diederichs, 1995).

FIGS. 3G and 4G show the result of the alignment between the Fv region(respectively of MNAC13 and αD11) and the tertiary structures of thealpha carbon atom skeletons of the humanized or human origin antibodies,selected on the basis of the optimal alignment of the primary structureswith the antibody to be humanized and of the high resolution of theavailable structural data.

To assess the degree of superposition of each individual structure, ofhuman origin or engineered, both with MNAC13 and with αD11 the RMS wascalculated between atoms of alpha carbon constituting the respectiveamino acid skeletons, not considering atom pairs with an RMS exceeding 2Å.

The selection of the optimal framework for humanization is configured asa three-variable problem, which can thus be represented in space, bothwhen associating the homology level and the degree of identity to thestructural alignment. This type of analysis was then conducted alsoreducing the regions in question in the two types of alignment to theregions of the respective frameworks.

As shown in FIGS. 3 and 4, the distributions of the antibodiesconsidered in the space of the three analyzed variables (respectively,value of RMS, percentages of atoms on which RMS was calculated and asimilitude index between primary structures, i.e. percentage of overallidentity -A-, of overall homology -C-, of identity at the frameworklevel -B-, of homology at the framework level -D-) are mutually coherentand consistent for both cases considered.

Moreover, comparing these distributions with the optimal position in thespace of the three variables which each antibody would occupy if it wereof human origin, it is possible clearly to identify the human originantibody that most approximates this ideal position at the primary andtertiary structure level. To rationalize, in the case of bothantibodies, this result, in each of the four analyses the deviationsfrom the hypothetical optimal position were calculated for each positionof the humanized or human origin antibodies considered (FIGS. 3E and 3Ffor MNAC13 and FIGS. 4E and 4F for αD11). In this case, too, the resultsare consistent and confirm the previous indications.

On the basis of this method of selection, two different humanizedantibodies were selected as acceptor framework in the subsequent processof CDR grafting for the humanization of the two antibodies neutralizingthe NGF/TrkA interaction. In particular, FIGS. 3H and 4H show thestructural alignment at the level of the Fv region of the two blockingantibodies with the respective selected humanized antibody, i.e. usingthe PDB, 1JPS codes for αD11 and 1AD0 codes for MNAC13. FIGS. 3I and 4Icompare the same region of the murine antibody with the model of thesame antibody following CDR grafting.

Once the CDRs are defined, the canonical classes (defined by Chothia andLesk) to which they belong were identified and subsequently thecanonical residues in the humanized antibody were maintained: for eachantibody, they were highlighted with underlined character in FIG. 5 andFIG. 6.

In regard to the subsequent analysis of the retro-mutations to beintroduced, to maintain the residues that mediate the interactionbetween the light chain and the heavy chain of the variable domains, thefollowing retro-mutations were inserted to maintain the interfacebetween the two domains:

-   L46 and H35, H37 for MNAC13-   L34, L46, L92 and H35 for αD11.

Moreover, to maintain the characteristics of the Vernier zone, thefollowing retro-mutations were made:

-   L98 for MNAC13

H71 for αD11 (which in any case regard substitutions for amino acidresidues represented in human consensus sequences).

Subsequently, following the comparison with the main consensus sequencesof human immunoglobulins, the following retro-mutations were made:

-   L1, L2, L13, L50, L73, L104 and H24, H48, H49, H73, H76, H82B, H87,    H90, H93 for MNAC13-   L56 for αD11

Moreover, again on the basis of the consensus sequences of humanimmunoglobulins, in the humanized form of MNAC13 the following mutationswere introduced which insert residues preserved in the human instead ofthe unusual residues present both in the donor and in the acceptorframework.

-   L42 (L→Q), L83 (I→V) and H83 (Q→R), H89 (I→V).

For the same reason, the following mutation was introduced in thehumanized form of αD11. H67 (V→F).

The respective pairs of crystallographic structures were modified, firsteffecting the grafting of the CDRs of animal origin in the humanizedframeworks. Then, all the mutations and retro-mutations described abovewere introduced. The modified structures were then assembled incomposite immunoglobulins. The resulting models were refined byminimizing mechanical energy (in terms of torsion angles and bindingangles and distances) using the force field.

Humanization of the MNAC13 and αD11 Monoclonal Antibodies

After selecting the donor humanized antibody of the framework to achievethe CDR grafting of MNAC13, the respectively variable regions aredesigned which combine the murine CDRs of MNAC13 with the framework ofthe humanized antibody modified according to the mutations set outabove. A similar procedure was followed for αD11. Substantially, the twohumanized variable regions can be obtained by a procedure based on theoverlap assembly PCR method, using oligonucleotides of about 80 bases,which alternate the sense and anti-sense filament with consecutivesuperposed for a length of 20 bases in such a way as to allow theformation of partially dual filament molecules as a result ofhybridation (FIGS. 7B and 8B). After filling discontinuities by means ofVent polymerase, the dual filament is amplified for PCR using as primerstwo short oligonucleotides bearing the sequences at the 5′ of the dualfilament itself together with restriction sites suitable for thesubsequent directional cloning (respectively ApaLI/BgIII for the cloningof the variable domain of the light chain and BssHII/BstEII for thecloning of the variable domain of the heavy chain), after digestion withrespective restriction enzymes, in the pVLexpress plasmid for thevariable domain of the light chain (FIG. 9A) and in the pVHexpressplasmid for the variable domain of the heavy chain (FIG. 9B). Thesecarriers allow to express in fusion with the cloned sequences theconstant domains of human origin respectively Cκ and CH1, CH2 and CH3.Using these vectors, it is therefore possible to express both antibodiesin the form of IgG1 molecules (FIGS. 9C and 9D1) in human cell lineslike those listed above. To obtain both humanized antibodies in the formof Fab fragments, it is sufficient to act solely on the carrier in whichthe heavy chain is cloned. In particular, it is possible to substitutethe entire constant part with the sole CH1 domain amplified for PCRusing specific primers provided with restriction sites at the extremesfor the SacII-XbaI directional cloning (as shown in FIG. 9D2).

Lastly, to obtain the MNAC13 humanized antibody in the form ofimmunotoxin, it is possible to express at the carboxy-terminal of theconstant domain CH1 the basic protamin protein amplified for PCR usingspecific primers provided with the restriction site at the extreme fornon direction Xbal cloning (as shown in FIG. 9D3).

Expression and Binding Assay of the MNAC and αD11 Humanized Antibodies

250 thousand COS cells were co-transfected with 1 .mu.g of codingplasmidic DNA for VH at VK of each humanized antibody (a total of 2 μg)by means of FuGENE according to recommended protocol (Roche). Theconstructs were used to obtain the humanized antibodies in the form ofIgG1.

In parallel to the co-transfections of the constructs described above,the corresponding chimeric forms were co-transfected for each antibody,i.e.:

-   the murine VH of MNAC13 cloned in CMV pVH express and the murine Vk    of MNAC13 cloned in CMV pVk express;in regard to αD11 the rat VH is    cloned in fusion with the C.gamma. of human origin in pcDNA1 and the    rat Vk is cloned in fusion with the C.gamma. of human origin pcDNA1.

After 72 hours from the transfection, the supernatant containing theimmunoglobulins expressed by the host cells was collected, andconcentrated using Centriprep 50 (Amicon).

The ability to recognize the respective ligands of the two humanizedantibodies was verified by means of ELISA assay and compared withrespective chimeric forms. The results are shown in FIGS. 10 and 11.

For immobilization on plastic, 96 well Maxi sorb plates were incubatedat 4° C. overnight with a solution containing the respective ligands ofthe two antibodies (the purified recombinant immunoadhesin TrkA Cameland the murine NGF purified from submandibular glands) at aconcentration of 10 μg/ml in 0.1M pH 9.6 sodium carbonate buffer.

After one hour of blocking with PBS containing 3% milk (MPBS) at ambienttemperature, concentrated supernatants were incubated with serialdilutions (1:2, 1.20; 1:200) and in parallel also with the supernatantof non transfected COS cells (negative control).

After incubation with the primary antibody (which recognizes theconstant C.gamma. region of human origin) and with the secondaryantibody (anti-rabbit conjugated with peroxidase), it is possible todetect the binding activity as optical density at 450 nm (OD450) bymeans of incubation with the TMB substrate (TECNA). The respectivemonoclonal antibodies at a concentration of 500 ng/ml were included aspositive controls.

FIG. 10B shows the results of a similar ELISA assay conducted afterpurification by means of affinity chromatography of the expressedimmunoglobulins.

In detail, the supernatants of the transfected cells were collected and,after removing cellular detritus by centrifuging, they were incubatedwith 100 .mu.l of Protein G Sepharose and after extensive washings withPBS each protein was eluted with 1 mM HCl and the pH was neutralizedimmediately after elution with 1 M Tris pH 8.8. The respectiveconcentrations were estimated with Lowry assay.

BIBLIOGRAPHY

-   Adair et al. Hum. Antibod. Hybridomas 5:41 (1994)-   Angeles et al. Anal Biochem; 236, 49 (1996)-   Baca et al. J. Biol. Chem. 272:10678 (1997) [0135]Baker et al.    “Antigen and Antibody Molecular Engineering”: 61 (1994)    [0136]Barinaga, Science; 264, 272 (1994)-   Benhar et al. P.N.A.S. 91:12051 (1994)-   Bentley et al. Nature; 348, 254 (1990)-   Benvenuto et al. Plant Mol. Biol; 17 865 (1991)-   Berardi et al. PNAS; 91, 684 (1994)-   Berman et al. Nucleic Acids Res.; 28, 235 (2000).-   Bird et al. Science; 242, 423 (1988)-   Bold et al. J. Neurochem.; 64, 2622 (1995)-   Buhler et al. Bio/Technology; 8, 140 (1990)-   Buchner and Rudolph, Bio/Technology; 9, 157 (1991)-   Brunger, Acta Cryst.; D54, 905 (1998)-   Carter et al. P.N.A.S. 89:4285 (1992)-   Carugo and Pongor Protein Science 10, 1470 (2001)-   Carugo, J. Appl. Cryst.; 36, 125 (2003)-   Cattaneo et al. J. Neurosci.; 19, 9687 (1999)-   Cesareni, FEBS Lett; 307, 66 (1992)-   Clackson et al. Nature; 352, 624 (1991)-   Chaudhary et al., Nature; 339, 394 (1989)-   Chen et al., Gene Ther; 2, 116 (1995)-   Chothia & Lesk, J. Mol. Biol.; 196, 901 (1987)-   Chothia et al. Nature; 342, 878 (1989).-   Co et al. PNAS; 88, 2869 (1991)-   Co et al. J. Immunol. 148:1149-1154 (1992)-   Cook et al. Prot. Engng. 9:623-628 (1996)-   Couto et al. “Antigen and Antibody Molecular Engineering” 55 (1994)-   Daugherty et al. Nucleic Acid Res. 19:2471 (1991)-   De Martino et al. Antibod. Immunoconj. Radiopharmaceut. 4:829    (1991).-   Delsite et al. J. Androl.; 17, 481 (1996)-   De Schryver-Kecskemeti et al. Arch. Pathol.; 111, 833 (1987)    [0165]Diederichs, Proteins: 23 187 (1995)-   Djakiew et al. Cancer Res.; 51, 3304 (1991)-   Domenici et al. Vis Neurosci.; 11, 1093 (1994)-   Durin et al. Plant Mol. Biol.; 15, 281 (1990)-   Eigenbrot et al. Proteins 18: 49 (1994)-   Ellis et al. J. Immunol. 155:925 (1995)-   Foote & Winter J. Mol. Biol. 224:487 (1992)-   Garrard et al. Bio/Techniques; 9, 1373 (1991)-   Gillman & Smith, Gene; 8, 81 (1979)-   Goretzki et al. Surgery; 102, 1035 (1987)-   Gorman et al. PNAS; 88, 4181 (1991)-   Graziano et al. J. Immunol. 155:4996 (1995)-   Gram et al. PNAS; 89, 3576 (1992)-   Geldof et al. J. Cancer Res. Clin. Oncol.; 123, 107 (1997)-   George et al. The Prostrate; 36, 172 (1998)-   Goding, Monoclonal antibodies: Principle and practise, 2nd edition,    Academic Press (1986)-   Gussow & Seemann Meth. Enzymol. 203:99 (1991)-   Hakimi et al. J. Immunol. 151:1075 (1993)-   Hamilton et al. J. Infect. Diseases 176:59 (1997)-   Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor    Laboratory (1988)-   Hiatt et al. Nature; 342, 76 (1989)-   Hood et al. Immunology, Benjamin, N.Y., 2nj ed. (1984)-   Hsiao et al. Prot. Engng. 7:815 (1994)-   Hunkapiller and Hood, Nature; 323, 15 (1986)-   Huston et al. PNAS; 85, 5879 (1988)-   Jancarik & Kim, Appl. Cryst.; 24, 409 (1991)-   Jones et al. Nature; 321, 522 (1986)-   Kashmiri et al. Hybridoma 14:461 (1995)-   Kettleborough, Protein Engineering; 4, 773 (1991)-   Kabat, Sequences of Proteins of Immunological Interest (National    Institutes of Health,-   Bethesda Md., 1987 e 1991) [0196]Kleywegt & Jones, Structure; 2,    1241 (1994).-   Kolbinger et al. Prot. Engng. 6:971-980 (1993)-   Kodandapani et al. Biochem. Biophys., Res. Comm.; 251, 61 (1998)-   Koizumi et al. Pathol. Int.; 48, 93 (1998)-   Lachyankar et al. Cancer Res; 57, 532 (1997)-   Lanzavecchia et al. Eur. J. Immunol.; 17, 105 (1987)-   Larrick & Fry, Hum. Antibodies Hybridomas; 2, 172 (1991)-   Laskowski et al. J. Appl. Cryst.; 26, 283 (1993)-   Lefkovits e Pernis, Immunological Methods, Vols. I and II (Academic    Press, N.Y., 1979 and 1981).-   Leger et al. Hum. Antibod. 8:3-16 (1997)-   Lewin & Mendell, Trends Neurosci; 16, 353 (1993)-   Lewis & Crowe Gene 101:297 (1991)-   Lindsay, Ciba Foundation Symposium; 196, 39, (1996)-   MacGrogan et al., J. Neurochem.; 59, 1381 (1992)-   Maeda et al. Hum. Antibod. Hybridomas 2:124 (1991)-   Maffei et al. J Neurosci; 12, 4651 (1992)-   Marchetti et al. Cancer Res.; 56, 2856 (1996)-   Matsushima & Bogenmann, Mol Cell Biol.; 13, 7447 (1993)-   Meade et al. Bio/Technology; 8, 443 (1990)-   McGregor et al. PNAS; 96, 4540 (1999)-   Miknyoczki et al. Int. J. Cancer; 81, 417 (1999)-   Miknyoczki et al. Crit. Rev. Oncogenesis; 7, 89 (1996).-   Miralles et al. J. Endocrinology; 156, 431 (1998)-   Molnar et al. Eur J Neurosci; 10, 3127 (1998)-   Molnar et al. Neuroreport; 8, 575 (1997)-   Monoclonal Antibodies for Cancer Detection and Therapy (eds. Baldwin    and Byers,-   Academic Press, 1985), 159, 224-   Muragaki et al. J Neurosci; 17, 530 (1997)-   Nakagawara et al. N Engl J Med.; 328, 847 (1993)-   Navaza Acta Cryst.; A50, 157 (1994).-   Oelmann et al. Cancer Res.; 55, 2212 (1995)-   Ohta et al. J. Pathol.; 181, 405 (1997)-   Oikawa, et al. Int. J. Pancreat.; 18, 15, (1995)-   Olsnes and Phil, Pharmac. There; 25, 355 (1982)-   Otwinowski & Minor, Methods Enzymol.; 276, 307 (1997)-   Paik et al. J. Nucl. Med.; 23, 37 (1982)-   Passaniti et al. Int. J. Cancer; 51, 318 (1992)-   Paul, Fundamental Immunology, 3rd ed. Raven Press, N.Y., (1993)-   Pflug et al. Mol. Carcin.; 12, 106 (1998)-   Pflug et al. Endocrinology; 136, 262 (1995)-   Pflug et al. Cancer Res.; 52, 5403 (1992)-   Poul et al. Mol. Immunol. 32:101 (1995)-   Presta et al. J. Immunol. 151:2623 (1993)-   Putlitz et al. Bio/Technology; 8, 651 (1990)-   Queen et al. P.N.A.S. 86:10029 (1989)-   Remington's Pharmaceutical Science (15th ed., Mack Publishing    Company, Easton, Pa., 1980)-   Revoltella & Butler, J. Cell. Physiol.; 104, 27 (1980)-   Riechmann et al., Nature; 332, 323 (1988)-   Roberts et al., Nature; 328, 731 (1987)-   Roguska et al. Prot. Engng. 9:895 (1996)-   Roguska et al. Prot. Engng. 9:895 (1996)-   Roguska et al. PNAS, 91:969 (1994)-   Rosok et al. J. Biol. Chem. 271:22611 (1996)-   Routledge et al. Eur. J. Immunol. 21:2717 (1991)-   Ruberti et al. J Neurosci.; 20, 2589 (2000)-   Ruggeri et al. Current Medicinal Chemistry; 6, 845 (1999)-   Sato et al. Canc. Res. 53:851 (1993)-   Sato et al. Mol. Immunol. 31:371 (1994)-   Sato et al. Hum. Antibod. Hybridomas 7:175 (1996)-   Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).-   Scott & Smith, Science; 249, 386 (1990)-   Sha and Xiang Canc. Biother. 9:341 (1994)-   Shearman et al. J. Immunol.; 147, 4366 (1991)-   Skerra et al. Bio/Technology; 9, 273 (1991)-   Sijmons et al. Bio/Technology; 8, 217 (1990)-   Sims et al. J. Immunol. 151:2296 (1993)-   Singer et al. J. Immunol. 150:2844 (1993)-   Swimmer et al. PNAS; 89, 3756 (1992)-   Tagliabue et al. J. Biol. Chem.; 275, 5388 (2000)-   Tempest et al. Prot. Engng. 7:1501 (1994)-   Tempest et al. Int. J. Biol. Macromol. 17:37 (1995)-   Tempest et al. Biotechnology; 9, 266 (1991)-   Thorpe et al. Monoclonal Antibodies in Clinical Medicine, Academic    Press, 168 (1982) U.S. Ser. No. 07/290,968-   Verhoeyen et al. Science 239:1534 (1988)-   Verhoeyen et al. “Monoclonal Antibodies”: 37 (1991)-   Verhoeyen et al. Immunol. 78:364 (1993)-   Winnacker, From Genes to Clones (VCH Publishers, N.Y., 1987)-   Winter and Milstein, Nature; 349, 293 (1991)-   Woolf et al., Neuroscience; 62, 327 (1994).-   WO91/08216, 1991 [0276]Zhu et al., J. Clin. Oncol., 17, 2419 (1999)-   Zhu and Carter J. Immunol. 155:1903 (1995)

1. A method for humanizing the VH and VL variable regions of an anti-NGFantibody of non-human origin, comprising the steps of: a) providing thecrystallographic structure of the VH and VL regions of the anti-NGFantibody; b) pre-selecting a series of 0 to n possible frameworksacceptors of human origin or humanized antibodies, whose structure wasdetermined experimentally with a resolution of no less than 3 Å, basedon the highest level of homology and identity with the primary sequenceof the framework of the anti-NGF antibody; c) conducting a structuralcomparison between the VH and VL variable regions of the anti-NGFantibody and the regions VH and VL obtained in b), respectively andcalculating for each comparison the RMS, to identify the region VH andthe region VL of human origin with the smaller RMS; and d) inserting inappropriate position the sequences of the regions CDR of the anti-NGFantibody in the human sequences identified in c). 2-24. (canceled)